Journal: Cerebral cortex (New York, N.Y. : 1991)

Article Title: Dysregulation of TrkB Receptors and BDNF Function by Amyloid-β Peptide is Mediated by Calpain.

doi: 10.1093/cercor/bhu105

Figure Lengend Snippet: Figure 3. Calpain cleaves TrkB downstream of Shc-binding site (Tyr515). (A) Ponceau S staining of a PVDF membrane after transfer from SDS–PAGE. Purified cytosolic domain of human TrkB (TrkB active aa.450-end, Millipore) was digested with purified m-calpain, producing TrkB-ICD. TrkB-ICD band was cut (dashed square) and submitted for N-terminal sequencing (Edman degradation). CaCl2 (2 mM) was present in all conditions. (B) Multiple alignment of TrkA, TrkB, and TrkC protein sequences for different species. The Shc-binding motif, the Trk kinase domain, and the calpain-cleavage site identified by N-sequencing are identified in the sequence. Protein sequences were obtained in UniProtKD, and the multiple alignments were performed using the Clustal Omega tool. The proline residue (P) present in the TrkB sequence (*) was not detected by N-sequencing. (C) Schematic

Article Snippet: The phospho-TrkA (Tyr 490) rabbit polyclonal antibody (1:1500), which detects TrkB receptor when phosphorylated on the corresponding residue (Tyr515), was from Cell Signaling Technology.

Techniques: Binding Assay, Staining, Membrane, SDS Page, Sequencing, Residue

Journal: Molecular Cancer Therapeutics

Article Title: Dual Targeting of CDK and Tropomyosin Receptor Kinase Families by the Oral Inhibitor PHA-848125, an Agent with Broad-Spectrum Antitumor Efficacy

doi: 10.1158/1535-7163.mct-10-0190

Figure Lengend Snippet: Figure 3. A, effect of PHA-848125 on phosphorylation status of pRb. A2780 cells were treated for 24 h with 1 and 3 μmol/L PHA-848125. Total cell lysates were immunoblotted and the different sites of phosphorylation were probed with specific antibodies. B, effect of PHA-848125 on several CDK-related proteins. A2780 cells were treated with 1 μmol/L PHA-848125 for indicated times. Total cell lysates were immunoblotted with specific antibodies. C, cell cycle distribution of A2780 cells treated with 1 μmol/L PHA-848125 was determined by flow cytometry at different time points and compared with the corresponding control cells. D, the same samples used for cell cycle evaluation were analyzed by immunocytochemical staining for BrdUrd incorporation to quantify DNA replication. E, cell cycle distribution of A2780 cells, untreated or treated with 1 and 3 μmol/L PHA-848125 for 24 h. F, effect of PHA-848125 on TRKA phosphorylation and downstream pathway. DU-145 cells were serum starved and pretreated with different doses of the compound for 30 min before NGF stimulation for 15 min. Total cell lysates were immunoblotted with specific antibodies.

Article Snippet: Immunoblotting was done according to ard procedures, and staining was done with the folg antibodies against: retinoblastoma protein (pRb), B1, cyclin D1, cyclin A, and Kip/p27 (Pharmingenosciences); Cdc6 (NeoMarkers); p53, Cdc25A, TRKA, hospho-TRKA (Santa Cruz Biotechnology); hospho-Thr821 and pRb-phospho-Ser249/252 (Bio); and pRb-phospho-Ser780, pRb-phospho-Ser807/811, ho-phospholipase Cγ, phospho-AKT, and total (Cell Signaling).

Techniques: Phospho-proteomics, Flow Cytometry, Control, Staining

Journal: Nature Communications

Article Title: Deep visual proteomics uncovers nociceptor diversity and pain targets

doi: 10.1038/s41467-026-71418-8

Figure Lengend Snippet: a Schematic workflow showing patch clamp electrophysiology and ultra-low input proteomics of cultured sensory neurons. b IB4 labeling in magenta (scale = 20 µm). Independently repeated in at least three cultures from three different mice with similar results. c Representative traces of indentation-evoked, mechanically-activated currents in an IB4 − peptidergic nociceptor and the absence of such currents in an IB4 + non-peptidergic nociceptor. d Percentage of mechanically sensitive IB4 − and IB4 + neurons. The number of neurons is indicated in the bars. ** represents p = 0.0027, two-sided chi-sq test. e Representative action potential (AP) traces recorded from an IB4 − and an IB4 + nociceptor, with corresponding quantification of AP threshold, half peak duration, and afterhyperpolarization of the measured APs. IB4 − : n = 19, IB4 + : n = 13. Each dot represents a single neuron. Filled dots in the IB4 + group represent the two mechanosensitive non-peptidergic neurons. Data were obtained from at least three biological replicates, both male and female mice. * represents p = 0.04, unpaired two-tailed Student’s t test. f Representative images of sensory neurons cultured and stained on laser microdissection (LMD)-compatible membrane slides prior to LMD and mass spectrometry analysis (scale = 20 µm). g Numbers of precursors and proteins identified from peptidergic (IB4 − /TrkA + ) and non-peptidergic (IB4 + /TrkA − ) nociceptors and mechanoreceptors. N = 3. Each dot represents a biological replicate from a male mouse. h Principal component analysis of peptidergic and non-peptidergic nociceptors and mechanoreceptors. i Dynamic range of protein abundance in the two nociceptor subsets. j Volcano plot showing pairwise proteomic comparison between nociceptor subsets with marker proteins highlighted (two-sided Student’s t test). k Pathway enrichment analysis based on t test differences between nociceptor subsets. The top ten pathways with a Benjamini-Hochberg false discovery rate (FDR) < 0.05 are shown. Cyan = peptidergic, magenta = non-peptidergic, black = mechanoreceptor. Error bars in all panels represent s.e.m with the mean as the center. Source Data are provided as a Source Data file. Parts of Fig. 1a, f created in BioRender. Coscia, F. (2026) https://BioRender.com/a39426f .

Article Snippet: Before staining, slides were blocked in antibody diluent solution: 0.2% (v/v) Triton X-100, 5% (v/v) donkey serum and 1% (v/v) bovine serum albumin in PBS for 1 h at room temperature before overnight incubation at 4 °C with 1:500 polyclonal anti-goat TrkA (R&D systems, AF1056) primary antibody.

Techniques: Patch Clamp, Cell Culture, Labeling, Two Tailed Test, Staining, Laser Capture Microdissection, Membrane, Mass Spectrometry, Quantitative Proteomics, Comparison, Marker

Journal: Nature Communications

Article Title: Deep visual proteomics uncovers nociceptor diversity and pain targets

doi: 10.1038/s41467-026-71418-8

Figure Lengend Snippet: a Schematic workflow of sensory neurons cultured in an “inflammatory soup” containing nerve growth factor, NGF, and phorbol 12-myristate 13-acetate, PMA (top), along with representative indentation-evoked currents from a control (cyan) and an inflamed (red) peptidergic neuron. b Percentage of mechanically sensitive control and inflamed peptidergic nociceptors (left). * represents p = 0.027, two-sided chi-sq test. The number of neurons is indicated in the bars, along with their corresponding current amplitude (right). c Percentage of control and inflamed peptidergic nociceptors with residual current. * represents p = 0.045, two-sided chi-sq test. d Percentage of mechanically sensitive control (magenta) and inflamed (red) non-peptidergic nociceptors. The number of neurons is indicated in the bars. Volcano plots showing the pairwise proteomic comparison between control and inflamed neurons in non-peptidergic ( e ) and peptidergic ( f ) nociceptors with top regulated proteins highlighted (two-sided Student’s t test). g Bar plots showing raw protein levels of B3GNT2 in the control and inflamed groups of peptidergic (TrkA + , n = 3) and non-peptidergic (IB4 + , n = 3) nociceptors. Each dot represents a single mouse. h Schematic workflow of the validation experiment by knockdown of B3gnt2 using siRNA prior to inflammation and patch-clamp electrophysiology of peptidergic neurons. i Percentage of mechanically sensitive inflamed peptidergic nociceptors transfected with control siRNA (red) and B3gnt2-targeting siRNA (gray). The number of neurons is indicated in the bars. *** represents p = 0.0003, two-sided chi-sq test. j Current amplitudes (left) and percentage of neurons with residual currents in inflamed peptidergic nociceptors transfected with control siRNA (red, n = 26) and B3gnt2-targeting siRNA (gray, n = 11). Error bars in all panels represent s.e.m with the mean as the center. Source Data are provided as a Source Data file. Parts of Fig. 4a were created in BioRender. Coscia, F. (2026) https://BioRender.com/a39426f .

Article Snippet: Before staining, slides were blocked in antibody diluent solution: 0.2% (v/v) Triton X-100, 5% (v/v) donkey serum and 1% (v/v) bovine serum albumin in PBS for 1 h at room temperature before overnight incubation at 4 °C with 1:500 polyclonal anti-goat TrkA (R&D systems, AF1056) primary antibody.

Techniques: Cell Culture, Control, Comparison, Biomarker Discovery, Knockdown, Patch Clamp, Transfection

Journal: Journal of pharmacological sciences

Article Title: Role of transient receptor potential vanilloid subtype 2 in lower oesophageal sphincter in rat acid reflux oesophagitis.

doi: 10.1016/j.jphs.2021.03.010

Figure Lengend Snippet: Fig. 3. (A) Double-labelling of TRPV2 with CGRP, nNOS, CD117, TrkA, and a-smooth muscle actin (SMA) in the muscle layer of normal and reflux oesophagitis rats lower oesophageal sphincter. Arrows indicate co-localization of TRPV2-immunopositive nerve fibres with each marker. Scale bars ¼ 50 mm (CGRP, nNOS, TrkA, and CD117) or 100 mm (a-SMA). Arrows indicate co-localisation of double-labelled nerve fibres. (B) Co-localisation percentage of nNOS or CGRP with TRPV2-immunopositive nerve fibres in the muscle layer of normal and reflux oesophagitis rats. Data are presented as means ± SEM, n ¼ 6 rats per group.

Article Snippet: Other molecules were detected by indirect staining with mouse anti-ED1 and ED2 antibody (1:1000, AbD Serotec, Oxford, UK), sheep anti-CGRP antibody (1:4000, Enzo Life Sciences, Farmingdale, NY, USA), mouse anti-nitric oxide synthase antibody (1:500, SigmaeAldrich, St. Louis, MO, USA), goat-anti trkA antibody (1:100, R&D Systems, Minneapolis, MN, USA), goat-anti CD117 antibody (1:800, R&D Systems), rabbit anti-PGP9.5 antibody (1:20000, Abcam, Cambridge, UK), mouse anti-alpha smooth muscle actin antibody [1A4] (Alexa Fluor® 488) (1:2000, Abcam, Cambridge, UK), mouse antiNeuN antibody (1:1000, Millipore, Temecula, CA, USA), mouseanti NF200 antibody (1:2000, SigmaeAldrich), IB4 fluorescein labelled (1:100, Vector Laboratories, Burlingame, CA, USA) and donkey anti-mouse IgG secondary antibody, Alexa Fluor® 594, antirabbit IgG secondary antibody, Alexa Fluor® 594, and donkey antisheep IgG secondary antibody, Alexa Fluor® 594 (1:400, Thermo Fisher Scientific, Waltham, MA, USA).

Techniques: Marker

Journal: Cancers

Article Title: Sensitization of Cutaneous Primary Afferents in Bone Cancer Revealed by In Vivo Calcium Imaging

doi: 10.3390/cancers12123491

Figure Lengend Snippet: Bone marrow afferents are medium-to large sized neurons expressing the nociceptive marker Na v 1.8 and lacking expression of proprioceptive marker parvalbumin. ( A ) Rostrocaudal distribution of all Fast Blue+ retrogradely labelled bone marrow afferents within the analysed ipsilateral lumbar dorsal root ganglia (DRG) (L2–L5). ( B ) Proportion of Fast Blue+ neurons within the Na v 1.8-cre expressing population of DRG neurons throughout lumbar L2–L5. ( C ) Proportion of bone marrow afferents based on soma size. ( D – E ) Representative images of lumbar 3 DRG immunostained for TrkA (green) and Fast Blue retrogradely traced (blue) femoral bone marrow afferents in combination with tdTomato expressing neurons (red) driven by ( D ) Na v 1.8-cre, ( E ) Tmem233-cre, and ( F ) Pvalb-cre. White arrows indicate double positive Fast Blue/TrkA neurons; yellow arrows indicate triple positive neurons. Scale bar = 100 µm.

Article Snippet: Incubation with primary antibody in blocking buffer was performed overnight at RT for TrkA (R&D Systems Inc. Minneapolis, MN, USA—AF1056, 1:1000).

Techniques: Expressing, Marker